Synergistic antimicrobic agents of two bromonitroalkyl n-phenylcarbamates

ABSTRACT

SYNERGISTIC ANTIMICROBIC COMPOSITIONS COMPRISING AS THE ACTIVE INGREDIENT A MIXTURE OF 2-BROMO-2-NITROBUTYLN-PHENYLCARBAMATE AND 2-BROMO-2-NITROBUTYL-N-(3,4-DICHLOROPHENYL)-CARBAMATE.

3,592,929 SYNERGISTIC ANTIMICROBIC AGENTS OF TWO BROMONITROALKYLN-PHENYLCARBAMATES Heinz Gunter Nosler, Monheim, Rhineland, and RichardWessendorf, Hilden, Rhineland, Germany, assignors to Henkel & Cie. GmbH,Dusseldorf-Holthausen, Germany No Drawing. Filed July 25, 1968, Ser. No.747,452 Claims priority, application Germany, Nov. 7, 1967, H 64,376;Dec. 7, 1967, H 64,694 Int. Cl. A01n 9/20 US. Cl. 424-204 9 ClaimsABSTRACT OF THE DISCLOSURE Synergistic antimicrobic compositionscomprising as the active ingredient a mixture of 2-bromo-2-nitrobutyl-N-phenylcarbamate and 2-bromo-2-nitrobutyl-N-(3,4-dichlorophenyl-carbamate.

STATE OF THE ART Many organic nitro compounds are known to possessantibacterial properties and examples of such compounds are aromaticnitro compounds, nitro-substituted pyridine derivatives, nitrofurfuralderivatives, many aliphatic nitro, dinitro and polynitro compounds. Ofparticular interest as preservatives have been aliphatic nitro alcoholsand brominated derivatives thereof due to their broad spectrum ofactivity. These compounds, however, have the disadvantage of showingsigns of decomposition after storage under atmospheric conditions for ashort time and they generally have only a low degree of activity whichis disadvantageous for impregnating wood and textiles for instance.

Known fungicides include nitro alcohols and esters of nitro alcoholswith monoand polybasic carboxylic acids but these products are onlyeffective against fungi and not against bacteria. Therefore, theirlimited spectrum is too restricted for general use as antimicrobicagents. Up to now, there has been no reliable known connection betweenchemical structure and antimicrobic activity. For example, Urbanski inNitro Compounds, Warsaw, 1964, page 449 et seq., discloses that ethyl4-chloro-4,4-dinitro butyrate and 2-bromo-Z-nitro-propanediol-1(1,3)have a high activity against micro-organisms while ethyl 4-bromo-4,4-dinitro butyrate and 2-chloro-2-nitro-propanediol- (1,3) aresubstantially devoid of any such activity.

In copending commonly assigned U.S. patent application Ser. No. 747,453filed on even date herewith and entitled Novel AntimicrobicCompositions, there are described novel antimicrobic compositions havingas the active ingredient, a nitroalkyl-N-phenylcarbamate of the formulaY, A wherein Y is selected from the group consisting of chlorine andnitro, n is a whole number from to 2 United States Patent 0 wherein Y, nand R have the above definitions. These compositions are elfective atrelatively low concentrations.

OBJECTS OF THE INVENTION It is an object of the invention to providenovel synergistic antimicrobic compositions having a high degree ofactivity against bacteria and fungi.

It is another object of the invention to provide novel synergisticantimicrobic compositions having a short kill time.

These and other objects and advantages of the invention will becomeobvious from the following detailed description.

THE INVENTION The synergistic antimicrobic compositions of the inventionare comprised of a mixture of Z-bromo-Z-nitrobutyl-N-phenylcarbamate and2-bromo-2nitrobutyl-N- (3,4-dichlorophenyl)-carbamate in a weight ratioof 1:4 to 4: 1, preferably 1:1. The synergistic increase in activity isevidenced by greater effectiveness against both gram negative and grampositive bacteria and fungi and also by a broader spectrum of activity.The synergistic activity is all the more surprising as no synergisticactivity has been observed with mixtures of nitro alcohols, their esterswith monoand polycarboxylic acids and nitroalkyl-N-phenylcarbamates.

The antimicrobic compositions of the invention may be in the form ofliquid, pasty or solid preparations such as aqueous suspensions,emulsions, solutions in organic solvents, oils, ointments creams,pencils, powders, soaps, toothpastes and mouthwashes which may be usedas cleansing agents, general and special skin-treatment agents and othercosmetic preparations. The synergistic combina tion of the twobromo-nitrobutyl-N-phenylcarbamates may be used with particularadvantage in antimicrobial cleansing, disinfecting and preserving agentsfor textiles, floors, hospital appliances and instruments, andindustrial establishments such as dairies, breweries, and laundries.

Due to their very good antimicrobic activity and improved spectrum ofactivity, the aforesaid synergistic combination of the twobromonitrobutyl-N-phenylcarbamates is also specially suitable for thepreservation of textile, leather and paper adjuvants, adhesives, paints,cutting oils and all kinds of cosmetic preparations. For this purpose,an addition of 0.005 to 0.2% by weight, especially 0.01% by weight, ofthe synergistic combination is recommended. Amounts as large as 5.0% ofthe corn position may be used, however.

In a modification of the antimicrobic compositions of the invention,synergistic compositions are comprised of an effective amount of acomplexing agent having a calcium carbonate binding capacity greaterthan 230 mg. per gm. of complexing agent in the Hampshire test and thetwo nitroalkyl-N-phenylcarbamates in a weight ratio of carbamate tocomplexing agent of 111000 to 50:1, depending upon the specificcomponents.

The complexing agent in the compositions of the invention having acalcium carbonate binding capacity of more than 230 mg. in the Hampshiretest may belong to varied classes of compounds such as polycarboxylicacids, hydroxy carboxylic acid, aminocarboxylic acids, phosphonic acidsand polyphosphonic acids and their alkali metal salts.

The Hampshire method for determining calcium carbonate binding capacityis described in the publication of the Hampshire Chemical Corporation ofJune 1960, Hampshire NTA Technical Bulletin, appendix, page A2. In themethod, exactly 2 gm. of powdery complexing agent are dissolved in 50cc. of distilled water after which the solution is neutralized andadmixed with 10 ml. of a 2% sodium carbonate solution. The pH isadjusted to 1112 and the solution is diluted to 100 ml. Then thesolution is titrated with a calcium acetate solution containing 44.1 gm.of calcium acetate monohydrate per liter until a distinct and lastingturbidity occurs. The calcium carbonand B were found by the plate testdiscussed infra and the results are given in Table I. This variation ofthe dilution test for chemical disinfectants set up by DeutscheGesellschaft fiir Hygene und Mikrobiologie has the advanate-bindingcapacity is determined according to the for- 5 tage of using a solidculture media instead of 'a liquid l culture media. Solid culture mediahave the advantage of cm calcium acetate solution 25IfJSIrnfgmagDiISdtZS easily discern the effectiveness, particularlyWelght pormon of complexmg agent The desired test concentrations wereprepared by mix- =mg. calclum Carb nat bound 10 ing specific amounts ofthe substance solutions of suitable per f complexing agentconcentrations with specific amounts of liquid bouillon or A th th nbeer-wort agars, in sterile petri-dishes. The amounts, measer 1 ca e ar1 1m1cro Comp 081 o S ured with a pipette, of the substance solutionswere a of the invention to obtain compositions having an extrememaximumof 01 to 1 m1 and the total volume in the 1y Short time comprises addmgto the synerglstlc mm petri-dishes after admixing with the culture mediaamounttures of 2-bromo-2-n1trobutyl-N-phenylcarbamate and 2- 9 ed to 10m1 P f f g gi if fi ljs After solidification of the culture media, itssurface In Welg m 10 0 l 3 e was inoculated with the test germsuspension in bouillon or 9 which Surpnsmgly Improves their or wort,which contained about 10 germs per ml. The actmty b f y' 2Q incubationtook place at 37 C. or at 30 C. in the incu- These sald anllmlcmblccmpsltlons are mmpnsed bator, and lasted 8 days when bacterial Candidaalbicans to by welght of an al.cohol Selected from the group wasemployed. When Epidermophyton Kaufmann Wolf colislstmg 9 ethanol i 150pmp anol 50% by was used, it lasted 21 days. The duration of incubationof weight of dimethylsulfoxide, 0.005 to 5% by weight of the 21 days forEpidermophyton Kaufmanmwolf was chosen W g' i zg i l i 3 2: 2 3; 25 toconform to the above standard test, because in the evalg i i f zf 9 aref nation of disinfectants against fungi of the epithelium, a y If e 8 1b 0 i substance is considered as suitable when the growth of g y su 0X16 an O y Welg 0 6 carthe fungi after predetermined duration of action isdei f 11 1 d d 1 layed by at least 21 days. Therefore, it wasascertained n t e o .Owmg P es are 6.56m Severa pre' which of thesubstance concentrations worked into the ferred embodiments toillustrate the lnventlon. I-lowever, culture media was just aboutcapable to arrest the growth it should be understood that the inventionis not 1ntended of the test germs completely This Value thus ascertainedto be hmlted to the speclfic embodlment' was indicated as thresholdconcentration. The tests were EXAMPLE I carried out in varyingconcentration intervals. 0 1 m 01 e of 2 brOm o 2 nitrobutan01 wasadmixed with When the tested antimicrobic composition did not con- 0 1ole of hen lisoc anate in anh drous benzene and tain alcohol, it wastested in a solution containing acetone. rgilxture hiatedyat reflux to 5hours After In the following tables, the concentration and thedestrucdistilling off the majority of the benzene, the residue was nontunes for the composmons tested are reported recrystallized from a 1:1mixture of petroleum ether and 40 TABLE I benzene to obtain2-bromo-2-nitrobutyl-N-phenylcarba- Inhibiting com in mate (agent A)having a melting point of 87 C. Sub Proggrl- Using the same procedure,Z-bI'OmO-Z-IIItIObUtYI-jN- Stance A Stance B A113 Tmgerm(3,4-dichlorophenyl)-carbamate (B) having a melting 4.1 St I I point of79 C. was prepared from 2-bromo-2-nitrobuta- 1 25 +5 up Ly 060mm mucusnol and 3,4-dichlorophenyl isocyanate. 121, 8 h} }Eschmcma w.

EXAMPLE II 321;; iii

25 111A ll The inhlblting concentrations of synergistic comblna- 25 50PiZZ- Z5 J;,Z,Z,ZL,, tions of the two bromom'trobutyl-N-phenylcarbamatesA TABLE II I Agent; mixture (A+B) 1:1

ngre- Ingredients in dient Sta. E. Pseudo- Can- Epidar- Aspcr- Pam'-composltion percent aureus coli menus dida mophyt. gillus cilliumIsopropanol 10 A A A A A A A 20 A A A A A A A Dimethylsulioxide (DMSO)40 A A A A A A A Agent mixture 0. 1 A A A A A A A 0.5 A A A A A A AIsopropanOl 10 A A A A A A A DMSO 40 Isopropano] 20 A A A A A A A DMSO40 Isopropapol 10 A A A A A A A Agent mixture 0. 1

Isopropanol 10 A A A A A A A Agent mixture. 0. 5

Isopropanol 20 A A A A A A A Agent 1mxture. 0. l

Isopropanol 20 10 5 5 A 5 A A Agent mixture 0. 5

DMSO 40 A A A A A A A Agent mixture 0. 1

TABLE II-Continued Agent mixture (A-H3) 1:1 Ingre- Ingredients in dientSta. E. Pseuda- Can- Epider- Asper- Penicomposition percent aureus colimonas dida 'mophg t. gillua cillium DMSO A A A A A A A Agent mixture 0.

40 A 10 10 A 0. 3 A A 0.1

10 40 0.5 0.5 0. 5 0.5 0.5 10 5 Agent mixture. 0. 5

Isopropanol DMSO 0. 5 0. 5 0. 5 1 0. 5 1O 5 Agent mixture 0. 1

Isopropanol 20 DMSO 40 0. 5 0. 5 0. 5 0. 5 0. 5 0. 5 0.5 Agent mixture0. 5

Norm-Statement of killing time in minutes. A=no killing in 10 minutes.Test temperature, 20 0.

TABLE III Agent mixtures A+B, 1:1 A+B, 4:1 A+B, 1:4 Ingredient Sta. Sta.Sta. Ingredients in composition percent aureus E. cult aureus E. coliaureus E. coli Isopropanol 20 A A A A A A Dimethylsulioxlde (DMSO) 10 AA A A A A 20 A A A A A A 30 A A A A A A Agent mixture 0. 5 A A A A A A20 A A A A A A 10 Isopropanol 20 A A A A A A DMSO 20 Isopropanol 20 A AA A A A DMSO 30 Isopropanol 20 10 5 10 10 10 5 Agent mixture 0. 5

DMSO 10 A A A A A A Agent mixture 0. 5

DMSO 20 A A A A A A Agent mixture 0. 5

DMSO 30 A A A A A A Agent mixture 0. 5

Isopropanol 20 DMSO 10 5 3 5 5 5 3 Agent mixture 0. 5

Norm-Statement of killing time in minutes. A=no killing time in 10minutes. Test temperature, 20 C.

TABLE IV TABLE IV-Continued Agent mixture Agent mixture Ingre- Ingredient A+B A-i-B A-l-B dient A-l-B A-I-B A-l-B Ingredient in compositionpercent 2:1 1:4 1:1 Ingredient in composition percent 2 :1 1:4 1 :1

Ethanol 10 A A A 10 20 A A A 042 3 3 1 Dimethylsulfoxide (DMSO) 40 A A A20 0.5 A A A 40 0.5 0.5 0.5

Agentmixture 0.5 10 A A A 40 NOTE.Statement of killing time in minutes.A=no killing time in 10 minutes. Test temperature, 20 C. 20 A A A 40 10A A A Agent mixture 0.5 As can be seen from Tables II to IV, the killtime 1s drastically shortened by the mixture of carbamates, the Ethanol20 10 ,10 10 Agent mixture 0.5 alcohol and drmethylsulfoxlde as comparedto the md1- vidual components alone any combination of two of the DMSO40 A A A Agent mixture 0.5 components which 1s completely unexpected.

Examples of complexing agents having a calcium carbonate bindingcapacity of more than 230 mg. per gm. of complexing agent are shown inTable V.

TABLE V Mg. of calcium Carbonate bound by Complexing agent 1 gm. ofagent Kl l-hydroxyhexane-1,1-diphosphonic acid 280 K-2a-aminoethane-a,a-diphosphonic acid 930 K-3u-aminopbenzyl-a,a-diphosphonic acid 1460 Aminotrimethylenephosphonicacid (Dequest 2000) 820 Ethylenediamine tetramethylenephosphonic acid860 Aminodimethylenephosphonic acid N-acetic acid 850 Iminodiacetic acidN-methylenephosphonic acid 540 Hydroxyethanediphosphonic acid 810Phosphonic acetic acid 270 Citric acid 328 K-4 Diethylenetriaminopentaacetic acid (DTPA) 275 K-S 1,2 cyclohexanediamine tetra acetic acid(CDTA) 285 K-6 Ethylenediamino-tetra acetic acid (EDTA) 402 K-7Nitrilotriacetic acid (NTA) 578 K-S GOOH COOH 3HNHCHzCH2-NH( JH OHChelDP OH 250 The complexing agents listed in Table VI have a calciumcarbonate binding capacity below 230 mg. per gm. in the Hampshire testand when used in combination with the carbamates in the test there wasno increase in antimicrobic activity.

TABLE VI Mg. of calcium carbonate per gm. Complexing agent: ofcomplexing agent Hydroxymethylphosphonic acid Mesoxalic acid monohydrate6 l-cysteinhydrochloride monohydrate 14 Glycolic acid 45 Tetra sodiumpyrophosphate 125 n-Hexylaminodimethylenephosphonic acid 160 Sodiumtripolyphosphate 214 Hexamethylenediamino-tetramethylene phosphonic acid220 ANTIMICROBIC COMPOSITIONS (A) Antimicrobial solution: Parts byweight 2bromo-2-nitrobutyl-N-phenylcarbamate l2-bromo-2-nitrobutyl-N-(3,4 dichlorophenyl)- carbamate Spirit dil ad 50(B) Antimicrobial salves:

( 1 2-Bromo-2-nitrobutyl-N-phenylcarbamate 0.5

2-Bromo-2-nitrobutyl N (3,4-dichlorophenyl-carbamate 0.5 Vaseline albaAd 100 (2) 2 bromo-2-nitrobutyl N phenyl-carbamate 0.5

2-bromo-2-nitrobutyl-N- 3 ,4-dichlorophenyl)-carbamate 0.5 Decyl esterof oleic acid 15 Colloid dispersion of 90 parts fatty alcohol of 16 to18 carbon atoms and 10 parts of sodium alcohol sulfate of 16 to 18carbon atoms 26 Water 58 Parts by weight (C) Antimicrobial cream: a a

Mineral oil 45.9 Beeswax 5 Spermaceti 6 Cetyl alcohol 1 Lanolin 1 Water4O Perfume 0.1 2-bromo-Z-nitrobutyl-N-phenylcarbamate 0.5

2-bromo-2-nitrobutyl-N-( 3,4 dichlorophenyl)- carbamate 0.5 (D)Antimicrobial powder: Parts by weigh2-bromo-2-nitrobutyl-N-phenylcarbamate 0.52-bromo-2-nitrobutyl-N-(3,4-dichlorophenyl)- carbamate Powdered talcumad 10 (E) Antimicrobial tooth powder:

Precipitated calcium carbonate 94.7

Powdered soap 5 2-bromo-2-nitrobutyl-N-phenylcarbamate 0.152-bromo-2-nitrobutyl-N-(3,4-dichlorophenyl)- carbamate 0.15 (F)Disinfectant hand wash paste:

Sodium lauryl sulfate 45 Monoethanolamide of coconut fatty acid 3 Finelyground pumice stone 48 Sodium salt of nitrilotriacetic acid 22-bromo-2-nitrobutyl-N-phenylcarbamate 12-bromo-2-nitrobutyl-N-(3,4-dichlorophenyl)- carbamate 1 (G)Antimicrobial scouring agent:

Dodecylbenzene sulfonate (WAS 30%) 22 Sodium sulfate 2 Finely groundpumice stone 7 Finely ground quartz powder 66 Nitrilotriacetic acid 22-bromo-2-nitrobutyl-N-phenylcarbamate 0.5 2-bromo-2-nitrobutyl-N-(3,4-dichlorophenyl) carbamate 0.5 (H) Antimicrobial washing agent:

Dodecylbenzene sulfonate 24 Sodium polyphosphate 44 Sodium sulfate 14.5Sodium silicate 6.5 2-bromo-2-nitrobutyl-N-phenylcarbamate 0.52-bromo-2-nitrobutyl-N-(3,4-dichlorophenyl)- carbamate 0.5 (I)Antiseptic shampoo:

Sodium lauryl ether sulfate (2728% WAS) 42 Diethanolamide of coconutfatty acid 5 28 B. glycerin 4 Colloid dispersion of parts fatty alcoholof 16 to 18 carbon atoms and 10 parts of sodium lauryl sulfate 15 Water60 (K) Shampoo:

2-br0mo-2-nitrobutyl-N-phenylcarbamate 0.0052-bromo-2-nitrobutyl-N-(3,4-dichl0rophenyl)- carbamate 0.005 Sodiumlauryl ether sulfate (27-28% WAS) 40 Diethanolamide of coconut fattyacid 6 Water 54 Parts by weight (L) Emulsion shampoo:

2-bromo-Z-nitrobutyl-N-phenylcarbamate 0.005 2-bromo-2-nitrobutyl-N- 3,4-dichlorophenyl) carbamate 0.005 Sodium lauryl sulfate (90% WAS)Diethanolamide of coconut fatty acid 3 Ethylenglycol stearate 2 Sodiumchloride 1 Water 83 (M) Shampoo with egg yolk:

2-bromo-2-nitrobutyl-N-phenylcarbamate 0.0 12-bromo-2-nitrobutyl-N-(3,4-dich1orophenyl)- carbamate 0.01 C C fattyalcohol sulfate mixture (40% WAS) 44 Liquid egg yolk 2 Sodium chloride0.3

Water 52.7 (N) Bubble bath:

2-bromo-2-nitrobutyl-N-phenylcarbamate 0.005 2-br0mo-2-nitr0butyl-N- (3,4-dichlorophenyl) carbamate 0.005 Sodium lauryl ether sulfate (27-28%WAS) 69 Diethanolamide of coconut fatty acid 5 Water (O) Antimicrobialsolution:

2-bromo-2-nitrobutyl-N-phenylcarbamate 0.252-bromo-2-nitrobutyl-N-(3,4-dichlorophenyl)- carbamate 0.25 Isopropanol20.0 Dimethylsulfoxide 30.0 Water 50.0 (P) Hand disinfectant:

2-bromo-2-nitrobutyl-N-phenylcarbamate 0.25 2-bromo-2-nitrobutyl-N- (3,4-dichlorophenyl carbamate 0.25 Ethanol 20.0 Dirnethylsulfoxide 30.0Sodium lauryl sulfate 7.5 Monoethanolamide of coconut fatty acid 0.5Nitrilotriacetic acid 2.0 Water 40.0 (Q) Disinfectant for instruments:

2-bromo-2-nitrobuty1-N-phenylcarbamate 0.25 2-bromo-2-nitrobutyl-N- 3,4-dichlorophenyl) carbamate 0.25 Isopropanol 20.0 Dimethylsulfoxide30.0 a-Aminoethane-a,u-diphosphonic acid 10.0 Water 40.0

(R) Antimicrobial tincture:

(Specifically against bacterially overlapping mycosis)2-bromo-2-nitrobutyl-N-phenylcarbamate 0.252-bromo-2-nitrobuty1-N-(3,4-dichlorophenyl)- carbamate 0.25

Ethanol 20.0

Dimethylsulfoxide Hydrocortisone 0.5 Water 40.0

Parts by weight (S) Antimicrobial spray:

2-bromo-2-nitrobuty1-N-phenylcarbamate 0.25 2-bromo-2-nitrobutyl-N- (3,4-dichlorophenyl) carbamate 0.25 Isopropanol 25.0 Dimethylsulfoxide20.0 Water 55.0 Propellent gas 100.0

Various modifications of the compositions and method of the inventionmay be made without departing from the spirit or scope thereof and it isto be understood that the invention is intended to be limited only asdefined in the appended claims.

We claim:

1. An antifungal and anti-bacterial composition consisting essetially of0.005 to 5.0% by weight of the total composition of a mixture of 2bromo-Z-nitrobutyl-N- phenylcarbamate and 2bromo-2-nitro-butyl-N-(3,4-dichlorophenyl)-carbamate in a weight ratioof 1:4 to 4:1 and an inert carrier.

2. The composition of claim 1 wherein the weight ratio is 1:1.

3. The composition of claim 1 also containing a complex former having acalcium carbonate binding capacity greater than 230 mg. per gram ofcomplex former in a Weight ratio of carbamate mixture to complex formerof 121000 to 50: 1, said binding capacity being calculated as 25 timesthe number of cc. of solution of 44.1 gm. of calcium acetate monohydrateper liter to titrate to turbidity a solution of 2 gm. of complex formerand 10 cc. of a 2% sodium carbonate solution diluted to cc. divided bythe weight portion of complex former.

4. The composition of claim 1 which also contains 5 to 30% by Weight ofan alcohol selected from the group consisting of ethanol and isopropanoland 10 to 50% by weight of dimethyl sulfoxide.

5. The composition of claim 4 containing 10 to 20% by weight of thealcohol.

6. The composition of claim 4 containing 20 to 40% by weight ofdimethylsulfoxide.

7. The composition of claim 4 wherein the alcohol is isopropanol.

8. The composition of claim 4 wherein the alcohol is ethanol.

9. The method of killing bacteria and fungi which comprises contactingfungi and bacteria with an effective amount of a composition of claim 1to destroy the bacteria and fungi.

References Cited UNITED STATES PATENTS 2,951,786 9/1960 Pullen et a1.424--300 3,253,904 5/ 1966 Harrison 260-471C 3,384,539 5/1968 Mocotte424-300 OTHER REFERENCES Crown Zellerbach, Product Information BulletinAug. 1961 pp. land 5.

ALBERT T. MEYERS, Primary Examiner V. D. TURNER, Assistant Examiner US.Cl. X.R. 424--211, 223, 300

